Asymmetrical Flow Field-Flow Fractionation (AF4)

At SOLVE:

Typical uses of AF4 at SOLVE is for size characterization of all types of samples having size above ~3 nm / 10 kDa. It is especially used for aggregate analysis and the testing of large polydisperse compounds with a tendency to aggregate or adsorb to surfaces. It is also commonly used as an orthogonal method to SEC. Lately it has also been increasingly applied to complex samples such as serum and plasma. At SOLVE we  use AF4 instrumentation from Wyatt and the channels are connected to UV, RI, MALS and FL detectors.

Principle:

Asymmetrical flow field-flow fractionation (AF4) sometimes more simply shortened field-flow fractionation (FFF) is a separation methodology which separate macromolecules and particles in solution according to differences in size. The separation is based on the macromolecules differential location in a flow under the influence of a field (in the case of AF4, a secondary flow). AF4 is commonly used in connection with detectors such as UV to monitor the amount of the size separated components as they are eluted from the channel.

Applicability:

AF4 is a method applicable to a very wide range of compounds, such as, proteins, nanoparticles, viruses and polymers. AF4 is applicable to compounds of molecular weight of ~ 10 000 g/mol and upwards to compounds of approximately 1 µm in size. Typical uses are for stability testing of proteins, aggregation detection, quality control, molar mass distribution determinations etc.

Pros

  • Very wide size range
  • High resolution
  • Quantitative (with UV or RI)
  • Low shear and surface area
  • Fractions can be collected

Cons

  • Less applicable for <10 000 g/mol components
  • Concentration changes during separation
  • Specialized equipment
  • Require some experience to operate well
  • Molar mass and Mw through the use of standards (unless MALS is used)